0 Comments
You are here: Home › Science Overview › Archive › ESI - Tandem MS Coupling for Chemoselective and Enantioselective Separations of Chiral Amino Acid Derivates

ESI - Tandem MS Coupling for Chemoselective and Enantioselective Separations of Chiral Amino Acid Derivates

Sep. 07, 2011
ESI - Tandem MS Coupling for Chemoselective and Enantioselective Separations of Chiral Amino Acid Derivates
ESI - Tandem MS Coupling for Chemoselective and Enantioselective Separations of Chiral Amino Acid ... more

The separation and identification of traces of D - amino acids (D-AAs) in the presence of huge excesses of L - amino acids is of ever increasing importance in the field of molecular biology. It has been shown that D-AAs play key roles in cellular processes of higher developed species. They act as biochemical switches for insulin production [1], as neuromodulators in the human cortex [2, 3] and are signal transmitters for the production and excretion of hormones [4]. For these purposes they are endogenously synthesized through enzymatic racemization [5]. However, many functions and inflictions of D-AAs in mammalian cellular processes still remain to be clarified. Therefore there is a strong need for powerful highly selective and sensitive methods in order to precisely analyze these molecules. We herein want to present a method that is based on the generation of iron (II) ferrocenyl propion amides of amino acids and the subsequent separation on a cinchona alkloid anion exchanger type chiral stationary phase. The combination of LC separation with the chemoselectivity of highly sensitive triple quadrupole MS detection profits from great synergism and allows us to simultaneously detect the D - enantiomers of most chiral proteinogenic amino acids. We herein want to present the application of our method to biological samples like urine, serum whole blood and cell lysate from HT29 (human colorectal cancer cell line) and rat liver.

Aims

  • 1D - chromatographic method to enantioselectively separate most chiral proteinogenic amino acids
  • Introduction of a lable to enhance chromatographic selectivity and sensitivity in MS/MS detection
  • Simple derivatization procedure to allow short reaction times and 100% analyte conversion
  • Method adaptable to biological samples

Sample Preparation

  • Mechanical cell pulping by potter
  • Addition of internal standard d4-(2,3,3,3)-L-Alanine
  • Centrifugation of cell fragments and unpulped tissue
  • Precipitation of proteins by icecold MeOH
  • Centrifugation of protein fraction
  • Derivatization of supernatant (45min @ 45°C)

LC-MS/MS Measurements
SFP derivatives generally follow the elution order of D - amino acids before L-AAs on the quinine anion echanger QN-AX CSP.

Elution order is reversed by using the pseudoenantiomeric quinidine QD-AX selector. Glycine, however elutes between L and D fractions on both columns. Underivatized molecules and such without acidic function elute with the chromatographic front and thereby are separated from the analytes. MS selectivity makes chemoselective differentiation of AAs feasible. LC selectivity enables separation of isobaric compounds like leucine and isoleucine as well as enantioseparation of all chiral proteinogenic amino acid except proline.

The CHN derivatization allows baseline separation of all 19 chiral proteinogenic amino acids. The general elution order is D before L on QN-AX. This trend is reversed for Asp. Enantiomers of different AAs elute stacked. The quinoline lable is highly fluorescent and thereby also enables LIF detection. In 1D - chromatographic separations MS detection introduces a second dimenson, which is necessary to distinguish various AAs from each other.

Conclusions
Extremely low noise in MS selected reaction monitoring SRM mode with LOD in the 100 fmol range Synergism of MS selectivity & chromatographic selectivity through labelling strategies all proteinogenic L - amino acids found in liver, cell lysate and urine with both methods D - amino acids identified and quantified (relative to L-AAs) SFP derivatization supports separation of matrix compounds from D-AA traces Low additive concentrations facilitate MS detection in both cases.

Roland J. Reischl
Wolfgang Lindner
University of Vienna
Department of Analytical Chemistry
Vienna, Austria

This poster war presented at HPLC 2011, 19 - 23 June in Budapest, Hungary.

Please click here to read it as a PDF.

 

 

Authors:
Roland J. Reischl
Wolfgang Lindner

Sign up for our free newsletter for more news like this.

Related Articles :

Keywords : 1D-Liquid Chromatography Biology chiral amino acid derivates Chromatography D-amino acid ESI HPLC 2011 HPLC 2011 Poster Poster Roland J. Reischl Separation Spectrometry Univeristy of Vienna Wolfgang Lindner

RSS Newsletter